These results showed that Lys-GO is a bifunctional adsorbent for the removal of dyes and metal ions, and excellent adsorption efficiency was obtained. The maximum adsorption capacities for MB dye and Cu2+ were 1679.1 mg/g and 186.9 mg/g at 35 °C, respectively. The kinetics of adsorption followed well the linear pseudo-second-kinetic model. The isotherm results indicated that MB adsorption can be described with the Langmuir isotherm model, while the adsorption of Cu2+ can be described with the Freundlich model. The excellent adsorption capacity indicated that the Lys-GO may be a promising adsorption material for the removal of environmental pollutants.In this paper, novel L-lysine-modified graphene oxide (Lys-GO) was synthesized through amidation. The morphological and structural properties of Lys-GO were characterized using infrared spectrometry, scanning electronic microscopy and X-ray photoelectron spectroscopy. The as-prepared Lys-GO material was systematically investigated in a series of batch adsorption experiments for the removal of methylene blue (MB) and copper ions (Cu Recently, graphene-based materials have drawn attention because of their huge specific surface area, extraordinary electronic transport properties and unique adsorption properties [x/3D-graphene hybrid material as an electrode material enhanced the efficiency of hydrogen-producing in a fuel cell [Graphene is a two-dimensional carbon material with honeycomb network and spoperties –3. Theseoperties , electrooperties , environoperties  and cataoperties . For exauel cell . Mo et auel cell , which cuel cell –11. A hiuel cell . Howeveruel cell –16.Recently, functionalized graphene materials have shown great potential as highly efficient absorbers for the treatment of environmental pollutants and wastewater purification –19. Howe2+).According to this analysis, we tried to synthesize a novel graphene material for the removal of heavy metal ions and organic dyes from wastewater. In 2013, Gao et al. developed an environmentally friendly approach to reduce GO with L-lysine , which s2 to form Lys-GO. GO is an acidic material, and the basic L-lysine can adjust the acid–base properties of GO. Lys-GO showed excellent adsorption capability for the removal of methylene blue (MB) and Cu2+ from simulated wastewater.Herein, L-lysine was attached to the surface of GO by amidation between –COOH and –NH−1. The strong peaks around 3438 and 1400 cm−1 are ascribed to absorbed water. After L-lysine was grafted onto GO, the CH/CH2 stretching vibration of the obtained Lys-GO material can be seen at about 2923 cm−1. More significantly, a new peak at 1574 cm−1 indicated N–H (amide-II bands). The peaks at 1208, 1574 and 836 cm−1 are attributed to characteristic amide bonds [Firstly, the obtained materials were characterized by FTIR analysis . For GO,de bonds ,23. TheyNext, the chemical states and components of the Lys-GO material were measured by XPS analysis . The fulFinally, the morphology of as-prepared graphene materials was characterized by SEM . GO exhi2+ adsorption was studied. The initial pH value as an important parameter may control the adsorption process, particularly the adsorption capacity, because the adsorption equilibrium changes with different pH values. Hence, the variation of MB and Cu2+ adsorption on Lys-GO under different pH conditions was investigated  + b (R2 = 0.94). To further investigate the adsorption process, the experimental adsorption kinetic data were analyzed using two conventional kinetic models (linear pseudo-first-order and linear pseudo-second-order).From The linear pseudo-first-order kinetic model can be expressed as:qe and qt are the adsorbed amounts (mg/g) of MB or Cu2+ at equilibrium and at different times t, respectively. κ1 (min−1) is the rate constant of a pseudo-first-order model of adsorption. The values of qe and κ1 can be calculated from the intercept and slope of the linear plot of ln(qe − qt) as a function of t.where The pseudo-second-order model includes all the steps of adsorption including external film diffusion, adsorption, and internal particle diffusion, and can be expressed as:qe and qt are defined as above and κ2 is the rate constant of the pseudo-second-order model of adsorption (g/mg/min). The slope and intercept of the linear plot of t/qt against t yield the values of qe and κ2.where qe,cal, κ1, κ2, and R2 from R2 for the linear pseudo-second-order model reached up to 0.999, which is much better than that of the pseudo-first-order model. Addtionally, The values of qe,cal were also in accordance with the experimental adsorption capacity  obtained from the pseudo-second-order model. These results indicated that the pseudo-second-order kinetic model may describe the adsorption of MB on graphene. The adsorption kinetic model of Cu2+ was very similar to that of MB on Lys-GO, and the linear pseudo-second-order kinetic model fitted the adsorption of Cu2+ on Lys-GO.The calculated values of The adsorption equilibrium isotherm is a key for describing the distribution of the adsorbate molecules between the liquid and the solid phase in equilibrium. Several mathematical models have been widely used to describe equilibrium states of the adsorption of molecules on surfaces. To the best of our knowledge, most of the adsorption systems were generally analyzed by the models of Langmuir or Freundlich. Therefore, the experimental data were also fitted by Langmuir and Freundlich isotherms in this work.The Langmuir isothermal linear equation is described asand the Freundlich isothermal linear expression is represented byCe (mg/L) is the equilibrium concentration of MB or Cu2+ in solution, qe (mg/g) is the amount adsorbed on Lys-GO, qmax (mg/g) is the maximum monolayer adsorption capacity on Lys-GO, KL (L/mol) is a coefficient related to the energy of adsorption and is expected to vary with temperature; KF (mol(1 − n)·Ln/g) and n are constants of the Freundlich isotherm related to the adsorption capacity and adsorption tendency, respectively.where 2+ on Lys-GO are given in KL and qmax) and Freundlich isotherm constants (n and KF) are listed in R2 = 0.9879. The value of R2 (0.7669) obtained from the Freundlich isotherm was much lower than that of the Langmuir isotherm. For the adsorption isotherms of Cu2+ on Lys-GO in 2+ adsorption capacity was approximately 156.91 mg/g, and R2 was equal to 0.6722 using the Langmuir model. As compared with the Langmuir model, the Freundlich model was more suitable for describing the adsorption equilibrium of Cu2+ on Lys-GO. Similar result of Cu2+ adsorption was reported in [The adsorption isotherms of MB or Cuorted in .2+ with the help of other materials has been extensively studied. The adsorption capacities of other adsorbents were compared in 2+ are much better than those of the other previously reported materials. The excellent adsorption performance of Lys-GO might be attributed to two factors: 1) Grafting L-lysine on the GO can improve the affinity for MB and Cu ions because more functional groups are available. 2) Strong π–π interactions and vacancy defects are good for enhancing the adsorption capacity of graphene materials. These results indicated that Lys-GO material has great potential application in treating wastewater.The removal of MB and Cu2+ from simulated wastewater. Compared to GO, the performance and adsorption capacity of the modified Lys-GO material was significantly improved. The maximum adsorption capacity for MB and Cu2+ was 1679.1 mg/g and 186.9 mg/g, respectively. The kinetics of adsorption followed well the linear pseudo-second-kinetic model. For the Lys-GO material, the isotherm results showed that MB adsorption fitted the Langmuir isotherm model, while Cu2+ fitted the Freundlich model. The adsorption capacity of MB and Cu2+ on Lys-GO prepared from GO was much better than that of other materials, such as GO/MgO NCs or GO with different defects. The functionalized graphene material may be a promising candidate for the removal of environmental pollutants.A novel amino acid–graphene composite material was synthesized using graphene oxide (GO) and L-lysine. The interfacial property of GO can be improved by the chemical modification. The as-prepared Lys-GO material as an adsorbent was systematically evaluated with respect to the removal methylene blue (MB) and Cu4, NaNO3, H2SO4 (98%) and HCl (36–38%) were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai China). The deionized (DI) water used throughout all experiments was purified to 18.2 MΩ·cm with the Millipore system.Graphite powder was purchased from Shanghai Huayi Company . KMnO2+ concentration was analyzed using atomic absorption spectrometry at 324.8 nm .Lys-GO was characterized by scanning electron microscopy  and X-ray photoelectron spectroscopy . Fourier-transform infrared (FTIR) spectroscopy was performed on a Perkin-Elmer model 2000 FTIR spectrophotometer using the Spectrum v. 2.00 software package. MB solution was analyzed using a UV spectrophotometer  by monitoring the absorbance changes at the wavelength of maximum absorbance (664 nm). Cu2 (20 mL) in the presence of 0.5 mL of dry N,N-dimethylformamide (DMF) in a 50 mL round-bottomed flask and heated to 70 °C for 24 h, using an absorption device of neutralization tail gas. After completion of the reaction, the solvent was evaporated at 100 °C and the solid was washed by anhydrous tetrahydrofuran (THF). The obtained product was reacted with L-lysine (2.0 mmol) in anhydrous DMF (20 mL) at 90 °C for 12 h. The L-lysine modified graphene sheets were obtained by filtration and washed by 5 wt % NaHCO3, deionized water, and ethanol to remove the unreacted amino acids, respectively. Finally, the sample was dried under vacuum at 50 °C. The whole route of synthesis is shown in Typically, GO (30 mg) was treated with SOCl2+ solution of known concentration were transferred in flask and shaken at four different temperatures . The solutions were collected at different time points (5–250 min), and then centrifuged at 12,000 rpm for 10 min. The liquids were analyzed for MB or Cu2+ concentration measurements. The obtained data in batch mode studies were used to calculate for each sample of Cu2+ or MB as follows:The adsorption as a function of the time was studied to obtain the kinetics information. 10 mg absorbent and 10 mL of MB or Cuqe is the equilibrium adsorption capacity of Cu2+ or MB on Lys-GO (mg/g), V is the sample volume (L), C0 and Ce are the initial and equilibrium concentration of Cu2+ or MB (mg/L) respectively, and m is the weight of Lys-GO (g).where 2+ solution with different initial concentrations was added to 10 mg Lys-GO, and shaken for 250 min at 303 K. The supernatant was collected by centrifugation. The concentration of remnant MB was measured by UV spectrophotometry, referring to a standard curve. The concentration of remnant Cu2+ was analyzed using atomic absorption spectrometry at 324.8 nm.To quantify the adsorption isotherms, 10 mL of MB or Cu


In the present work, a novel infrared-assisted extraction coupled to headspace solid-phase microextraction (IRAE-HS-SPME) followed by gas chromatography-mass spectrometry (GC-MS) was developed for rapid determination of the volatile components in green tea. The extraction parameters such as fiber type, sample amount, infrared power, extraction time, and infrared lamp distance were optimized by orthogonal experimental design. Under optimum conditions, a total of 82 volatile compounds in 21 green tea samples from different geographical origins were identified. Compared with classical water-bath heating, the proposed technique has remarkable advantages of considerably reducing the analytical time and high efficiency. In addition, an effective classification of green teas based on their volatile profiles was achieved by partial least square-discriminant analysis (PLS-DA) and hierarchical clustering analysis (HCA). Furthermore, the application of a dual criterion based on the variable importance in the projection (VIP) values of the PLS-DA models and on the category from one-way univariate analysis (ANOVA) allowed the identification of 12 potential volatile markers, which were considered to make the most important contribution to the discrimination of the samples. The results suggest that IRAE-HS-SPME/GC-MS technique combined with multivariate analysis offers a valuable tool to assess geographical traceability of different tea varieties. Tea is one of the most popular beverages in the world owing to its attractive aroma, taste, and health benefits , 2. AccoAroma is one of the important indicators for the quality evaluation of teas . The comThe volatile components represent only about 0.01% of the total dry weight of tea, but they play an important role in determining the flavor of the products due to their low threshold value and high odor sensibility . Up to dMicrowave assisted extraction (MAE) is a relatively new extraction technique and has been widely used in food and natural product analysis, due to its special heating mechanism and good performance. It consists of electric field and magnetic field which oscillates perpendicularly to each other in frequency ranged from 0.3 to 300 GHz. However, the microwave radiation should be handled carefully because the leaked microwaves are dangerous to human health , 24.As an important form of electromagnetic wave, infrared (IR) radiation has wavelengths ranging from 750 nm to 1 mm, lying between visible radiation and microwaves . By matcIn the present work, infrared-assisted extraction coupled to headspace solid-phase microextraction (IRAE-HS-SPME) has been developed for the rapid analysis of the volatile constituents in green tea based on GC-MS. To our best knowledge, this is the first report of exploring IRAE-HS-SPME on the aroma composition in green tea. The extraction parameters such as fiber type, sample amount, IR power, extraction time, and IR lamp distance were investigated by orthogonal experimental design to acquire the optimal analysis conditions. A comparison between IR radiation and conventional water-bath heating was conducted. Multivariate statistical techniques such as PLS-DA, HCA were implemented to understand the potential characteristics according to the geographical origins of the samples. The application of a dual criterion based on variable importance in projection (VIP) scores and the results of one-way univariate analysis (ANOVA), allowed the identification of potential volatile markers from different geographical origins.In total, 21 commercially available roasted green tea samples were purchased from the local market. The detailed name and sources were listed in The SPME manual holder and the fibers of polyacrylate , polydimethylsiloxane-divinylbenzene , and carboxen-polydimethylsiloxane  were purchased from Supelco, USA. The n-alkane mixtures, consisting of C7–C40 straight-chain alkanes, were supplied by Supelco, USA.The infrared-assisted extraction system is shown in Prior to use, the fibers were conditioned by inserting them into the GC injector port according to the manufacture instructions: 0.5 h at 280 °C for PA, 0.5 h at 250 °C for PDMS-DVB, and 1 h at 300 °C for CAR-PDMS, respectively. Before sampling, each fiber was reconditioned for 5 min in the GC injector port at 250 °C to eliminate the possible remains on the fiber coating.The sample (0.5 g) was quickly introduced into 20-mL headspace vial and the vial was hermetically closed by a teflon/silicone septum. The needle coated with different fibers was inserted into the vials by manually penetrating the septum and the fibers were exposed to the headspace above the samples. After a period of extracting time at setting infrared power, the needle was removed from the headspace vial and directly inserted into the injection port of GC-MS. The effects of different fiber types , sample amount , IR power , extraction time , the distance between the IR lamp and the headspace vial  were investigated.The sample (0.5 g) was quickly introduced into 20-mL headspace vial and the vial was hermetically closed by home-made septum and immersed in a water bath. The needle coated with CAR-PDMS fiber was inserted into the vial by manually piercing the septum and the fiber was exposed to the headspace above the samples. After 20 min of extracting time at equality temperature of 90 °C, the needle was removed from the headspace vial and directly inserted into the injection port of GC-MS. The analytes were thermally desorbed in the splitless mode at 250 °C for 5 min.GC-MS was performed with an Agilent Technologies 7890A GC system coupled to an Agilent Technologies 7000C Triple Quadrupole mass spectrometer. Samples were analyzed on a HP-5ms (30 m ×I.D. 0.25 mm × film thickness 0.25 μm). Analytical conditions were as follows: The oven temperature was held at 40 °C for 0 min, and raised to 190 °C for 2 min at 3 °C/min, then increased to 290 °C at 10 °C/min, maintaining for 3 min; Splitless mode was conducted; Helium (99.999% purity) was used as the carrier gas at a rate of 1 mL/min.The mass spectrometer was operated in an electron-impact (EI) mode. The scan range was 50–450 m/z. The scan rate was 0.2s/scan. The temperatures at ionization source and interface were 230 and 280 °C, respectively.The qualitative analysis of the volatile components was processed by Agilent MassHunter Workstation Software Unknowns Analysis with the ability of peak picking, peak deconvolution and mass spectra comparison. The compounds were identified by comparing the obtained spectra with those of reference compounds from the National Institute of Standards and Technology (NIST14) and by the Kovàts retention indices calculated for each peak with reference to the n-alkane standards (C7–C40) running under the same conditions. Peaks were assigned when the similarity was above 80%. Any known artificial peaks were excluded from the data set. The relative percentages of various components in the samples were obtained by peak area normalization.Significant differences among green tea samples were determined by one-way ANOVA using SPSS 18.0 . The difference between two related results was considered to be statistically significant with values of p<0.05. HCA was also performed using the SPSS 18.0 . PLS-DA was conducted by SIMCA-P software with the version 11 . All variables were scaled with unit variance (UV) prior to PLS-DA.PLS-DA was conducted to develop models to find a two dimensional plane (discriminating plane) in which the tea samples (projected observations) on the PLS components were well separated according to their volatile compounds. As for the PLS weight plot, composition variables of weight plot can reveal the variables (specific volatile compounds) contributing to the separation.HCA is an unsupervised chemometric technique that reveals the natural groupings existing between samples characterised by the values of a set of measured variables. The results achieved were described by a dendrogram representing in a tree structure. In the present work, the similarities between samples were calculated on the basis of the Squared Euclidean distance, whereas Ward’s method was used as a linkage procedure to establish the clusters.The extraction process of IRAE-HS-SPME was affected by multiple parameters. In order to acquire the maximum extraction efficiency, a number of experiments under different conditions were performed. The effects of fiber type, sample amount, infrared power, extraction time, and the IR lamp distance on the extraction efficiency were investigated by orthogonal experimental design.The fiber coating is the “heart” of the extraction. In order to evaluate the selectivity of different fiber coatings, three types of fibers  with different polarity and inner structure were tested for the extraction efficiency of the volatile compounds in green tea. The total peak areas using PA and PDMS-DVB fibers were 76.66% and 89.72% of that using CAR-PDMS respectively see , indicat4) matrix was conducted to optimize the sample amount , extraction time , IR lamp power , and IR lamp distance   see . CompareIn this study, the orthogonal experiment design was used to evaluate the inner relationship and the influence sequence among factors, which might significantly affect the extraction efficiency. The Ki data was obtained by averaging the total peak area in the same level of each factor. The ki data was obtained by Ki data divided by the level number. The R value for each factor was calculated by finding the difference between maximum and minimum k value. The larger the R value for a factor, the stronger is the influence of the test factor on the result. The results suggest that the order of influence for extraction efficiency is RC > RD > RB > RA. In other words, the IR lamp power has the most significant impact on the detected amounts of volatile compounds in green tea followed by IR lamp distance, extraction time, and sample amount.The variation trend of ki can be used for determining the optimal level. From the results in Overall, the optimized extraction conditions were as follows: CAR-PDMS fiber, sample amount of 0.5 g, extraction time of 20 min, IR power of 175 W and IR lamp distance of 6 cm.On the basis of the optimized conditions, IRAE-HS-SPME was applied to identify the volatile profile of green tea samples from Hangzhou and Ya’an district. The volatile components were identified by comparing the obtained mass spectra with standard ones from NIST Mass Spectral library and by the Kovàts retention indices calculated for each peak with reference to the normal alkanes C7-C40 series. The relative amounts were calculated by the individual peak area relative to the total areas. The GC-MS chromatograms of green teas from different geographical origins were mapped in δ-cadinene, hexadecane, and cadalene. Saturated hydrocarbons are considered to have little contribution to tea flavor, while, unsaturated hydrocarbons play an important role in the flavor of tea [A total of 14 saturated and 13 unsaturated hydrocarbons were identified in 21 batches of green teas, accounting for 26.81% and 24.07% in Hangzhou and Ya’an district, respectively. There were significant differences (P<0.05) in the content of hydrocarbon compounds, such as α-terpinene, undecane, pentadecane, calamenene, r of tea . For exaThe results indicated that aldehydes were present with high proportion in green tea, representing 31.29% and 28.98% in Hangzhou and Ya’an district, respectively. Significant differences (P<0.05) in the content from the two regions were observed, including (E)-2-heptenal, benzaldehyde, -2,4-heptadienal, decanal, β-cyclocitral, and (E)-2-nonenal. The aldehydes, originated from thermal Strecker oxidative degradation of amino acids and fatty acids, play an important role on the entire odor because of their relatively low odor threshold values [Among the 11 identified ketones, 1-hepten-3-one, cis-jasmone, β-ionone, were detected as the major ketones, providing a special floral and woody odor. In particular, β-ionone, which can be produced either by enzymatic reactions during fermentation or by thermal degradation, is a significant contributor to the flavor of green tea due to low human odor perception thresholds . Other cAs far as alcohols are concerned, 1-octen-3-ol, benzyl alcohol, 1-octanol are the most abundant. These results are different from the previous reports , which cA total of 9 nitrogen compounds were identified. Caffeine, which is related to the taste of tea but contributed little to the aroma of tea, is most abundant for all green teas, comprising14.54% and 15.44% for Hangzhou and Ya’an district, respectively. Indole with typical flowery fragrance, is assumed to contribute the overall green tea odor. Pyrazines, such as 2,5-dimethylpyrazine, 3-ethyl-2,5-dimethylpyrazine, which are reported to be formed by Maillard reaction through Strecker degradation, contribute desirable roasty, sweet, and nutty odors .As for the rest of the identified compounds, methyl salicylate, which has a holly oil herbal fragrance, was found to be an important aroma compound. In addition, 2-pentyl-furan and 2-(2-propenyl)-furan with a burnt and sweet odor were identified in the present study . No signTo evaluate the extraction efficiency, IRAE was compared with conventional water-bath heating method. There are 53 components identified in green tea by water-bath heating see . The che9 while the total peak area obtained by water-bath heating is 6.23×109 (see -1) which can excite the vibrations in molecules in the modes of stretching, bending, rocking and twisting [-1. Additionally, most absorption peaks of volatile components also fall into the range of the IR radiation. That is to say, the wavelength of the infrared radiation matches the absorption characteristics of the active compounds in green tea. In addition, IR radiation owns high penetration capability, it heats the green tea sample in a three-dimension manner without heating the surrounding air, while a finite period of time is needed to heat the vessel before the heat is transferred to the sample in conventional heating mode [It is noteworthy that the IRAE contains most of the volatile components identified by water-bath heating. Moreover, the IRAE-HS-SPME has much higher extraction efficiencies than water-bath heating. Under the conditions investigated, the total peak area obtained by IRAE-HS-SPME is 15.01 ×10×109 see . The restwisting . The preing mode ,36. TherThe repeatability of the IRAE-HS-SPME method was determined by performing six replicate analyses on HZ-10 green tea sample under the optimum conditions. Ten frequent volatile components in green tea were selected. For these components, the relative standard deviations (RSDs) of retention time and peak area in green tea sample were calculated. As indicated in Above all, the IRAE-HS-SPME method is a rapid, high-efficient, solvent-free method for analysis of the volatile components in green tea. It is therefore considered that this method is promising and can be a good alternative to the traditional techniques.Multivariate analysis was implemented on the whole data consisting of a 21×82 matrix. The columns represent the green tea samples analysed and the rows represent the relative contents of the volatile metabolites determined by IRAE-HS-SPME. In particular, two classification models were built, in order to characterize the differences related to the geographical origins of the samples.PLS-DA is a multivariate technique used to classify different groups of samples. It is based on linking two data matrices, X (explanatory dataset) and Y (explicative dataset). In this study, two types of samples  were processed using PLS-DA method. As shown in PLS-DA was further performed to display specific volatile components that better explain the differences of the two regions. The results of PLS-DA highly coincide with the comparison result of the concentration. For example, some volatile compounds such as (Z)-3-hexenyl hexanoate (63), n-valeric acid cis-3-hexenyl ester (50), 2,5-dimethylpyrazine (5), α-terpinene (19), -2,4-heptadienal (15) and (Z)-3-hexenyl butanoate (43) presenting higher concentrations in Hangzhou samples than in Ya’an samples see  are stroSubsequently, the variable influence on the projection (VIP) parameter was used to select the metabolites which exhibited significant contribution in discriminating the two groups in PLS-DA model. Indeed, the VIP score is an index accounting for the relative importance: the higher the value of the VIP score, the more relevant the variable. Moreover, the VIP scores are normalized in a way that their average value on a particular model is always 1, so that a “larger than 1” criterion can be adopted to assess the significance of the contributions of individual predictors . In the HCA is an ideal technique for the crude classification of tea samples based on the contents of volatile components because it does not require previous information of test samples . HCA of δ-cadinene (74), hexadecane (78), indole (53), 1-(1-cyclohexen-1-yl)ethanone (2), α-terpinene (19), -2,4-heptadienal (15), β-cyclocitral (48) and 2,5-dimethylpyrazine (5) are the main compounds for discriminating between the cultivation systems. Among them, benzyl alcohol, α-terpinene and -2,4-heptadienal plays an important role in the flavor of green tea. We should remark that our study is based on two widely known cultivars. Further studies should be carried out with a higher number of cultivars to correlate the same behaviour in different systems.The application of a dual criterion, based on the univariate and multivariate analysis, allows the identification of potential volatile markers for different regions . In our In the present work, a novel IRAE-HS-SPME followed by GC-MS was developed for rapid determination of the volatile components in green teas. The optimal IRAE-HS-SPME performance was obtained under operating conditions: CAR-PDMS fiber, sample amount 0.5 g, extraction time 20 min, IR power 175 W and IR lamp distance of 6 cm. A total of 82 volatile components were extracted and identified in 21 green tea samples by the proposed technique. Multivariate technique including PLS-DA and HCA were successfully employed to provide a visual comparison and highlight the differences in the volatiles profiling of green teas from different geographical origins. Furthermore, 12 potential volatile markers were identified to be the most important variables in distinguishing the samples based on the variable importance in the projection values (VIP > 1.50) and on the category from one-way ANOVA (P<0.01). The results shows that the combination of IRAE-HS-SPME/GC-MS technique to multivariate analysis offers a valuable tool to assess geographical traceability of different green tea samples.S1 Fig(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.


The major drawback for the more widespread use of paclitaxel and its precious precursor, 10-deacetylbaccatin III (10-DAB III), is that they require large-scale extraction from different parts of yew trees (Taxus species), cell cultures, taxane-producing endophytic fungi, and Corylus species. In our previous work, a novel online two-dimensional heart-cut liquid chromatography process using hydrophilic interaction/ reversed-phase chromatography was used to introduce a semi-preparative treatment for the separation of polar (10-deacetylbaccatin III) and non-polar (paclitaxel) taxanes from Taxus baccata L. In this work, a combination of the absorbent (Diaion® HP-20) and a silica based solid phase extraction is utilized as a new, efficient, and cost effective method for large-scale production of taxanes. This process avoids the technical problem of two-dimensional preparative liquid chromatography. The first stage of the process involves discarding co-extractive polar compounds including chlorophylls and pigments using a non-polar synthetic hydrophobic absorbent, Diaion® HP-20. Extract was then loaded on to a silica based hydrophilic interaction solid phase extraction (silica 40-60 micron). Taxanes was eluted using a mixture of water and methanol at the optimized ratio of 70:30. Finally, the fraction containing taxanes was applied to semi-preparative reversed phase HPLC. The results revealed that using this procedure, paclitaxel and 10-DAB III could be obtained at 8 and 3 times more, respectively than by the traditional method of extraction. There is no other naturally occurring defense agent against cancer that has a stronger effect than paclitaxel, commonly known under the brand name of Taxol Tests determined that taxotere has excellent activity better than paclitaxel in some assays and were therefore developed as a parallel drug to paclitaxel , first introduced by Alpert, is an efficient technique used for the separation of polar and basic compounds because of its complementary selectivity against RP-HPLC , meaningBased on a previous study on production and purification of important taxanes including paclitaxel and 10-DAB III -24, SPE In this work, an offline combination is introduced using an absorbent for preliminary purification of taxanes. This is followed by a hydrophilic interaction solid phase extraction (SPE) based on a silica stationary phase. Consequently, a taxane containing fraction was eluted by an aqueous-organic mobile phase. Subsequently, this fraction was applied on a semi-preparative reversed-phase HPLC. Plant materials Taxus baccata L. gathered in June 2011 from the Botanical Garden of the University of Tehran, Iran. These Taxus needles were air-dried at room temperature. Stems were cut from plants, and mature needles were stripped manually from the stem, powdered and kept at -20 °C pending the extraction process. The investigation was performed on needles of ® HP-20 column followed by hydrophilic interaction SPE for obtaining polar and non-polar taxanes (paclitaxel and 10-DAB III).Chemicals n-hexane, dichloromethane were provided from Merck Co. . HPLC-grade acetonitrile was obtained from Caledon Co. . Ultrapure water was obtained from a Milli-Q water system. Diaion® HP-20 was purchased from Sigma-Aldrich® (3050 Spruce Street saint louis MO 63103 USA) with Product number of 13608 SupelcoTM. Celite 521 (from Acros Organic with CAS number: 61790-53-2) was received as a gift from the Pasteur Institute of Iran.Paclitaxel standard (98%) was purchased from Sigma Co. . The chemicals methanol, ethanol, Liquid-liquid extraction (LLE) n-hexane (2 × 10 mL). The hexane extract that contained lipids, waxes and pigments was discarded and the aqueous layer was extracted with dichloromethane (5 × 10 mL). During this stage the polar compounds such as carbohydrates remained in the aqueous phase and taxoids were extracted to the organic phase. Dichloromethane extracts were combined, evaporated under reduced pressure and the residue was dissolved in 15 mL acetonitrile.LLE was carried out according to Glowniak and Mroczek (18). An aliquot of 10 mL of methanol extract was mixed with 10 mL water and extracted with Instrumentation -1 was applied. The maximum absorption wavelength was 227 nm, and 20 µL of the sample was injected.Analytical separation was performed on an Agilent 1200 Series HPLC  fitted with variable-wavelength UV-Vis absorbance detector. Determination was performed using a C18 analytical column  and an isocratic elution with a mixture of acetonitrile and water at the ratio of 45/55 for paclitaxel and 30/70 for 10-DAB III. The flow rate of 1 mL minSemi-preparative purification was performed on a semi-preparative Knauer equipment including a WellChrom preparative pump K-1800, ultraviolet (UV) detector K-2501, a Büchi fraction collector B-684 , and YMC-Pack ODS-A . Separation of taxanes was carried out using acetonitril: water at the ratio of 45/55 for paclitaxel and 30/70 for 10-DAB III. Levels of flow rate, maximum wavelength and injection volume were 8 mL/min, 227 nm and 8 mL, respectively.TM LCQ TM DECA instrument, comprising an ion trap. An ionization device was used for sample analyses . The Xcalibur 2.0 SR2 software (copyright Thermo Electron Corporation 1998–2006) was used.Mass spectra were acquired by a Finnigan Silica based SPEIn this work, 10 g of the underivatized silica (40-60 micron particle size) was packed in to a 12 mL polypropylene column (13 × 2.5 cm). After loading the sample, column was washed with 500 mL deionized water and was subsequently washed with 500 mL of solvent mixtures from different water/methanol ratios. Absorbent treatment® HP-20, it was soaked and swelled in 100% (v/v) methanol for 12 h prior to use in order to omit impurities and the solvent was removed through washing with an adequate amount of distilled water. The washed wet resin was then weighed and used. After removing the major pigments and chlorophylls by the use of n-hexane, the methanolic extract was dried (Głowniak and Mroczek 1999) and suspended in 100 mL water. Resulting aqueous suspensions were treated by an absorbent in both column and dispersive modes . In the dispersive mode, absorbent was added directly to the extract. After 10 min stirring, absorbents were collected and then washed using 300 mL methanol. After evaporation of the solvent, extract was loaded on to a hydrophilic interaction column. The same procedure was applied in cases of column mode except that the resulting aqueous extract was applied to a cartridge packed with the absorbent. After loading of the sample, the column was washed with methanol. Fraction containing taxanes was dried using rotary evaporator under reduced pressure and dissolved in 15 mL acetonitrile.Absorbents in these tests, in both cases were used either as a dispersed agent or as a column. In the case of Diaione.g. 10-DAB III) and nonpolar (e.g. paclitaxel) taxanes . Then the column was eluted using different percentages of methanol. There are some reports about the application of Diaionproducts , 27. In ® HP-20 both as a dispersed agent and a column.Using methanol (100%) as eluent leads to elution of all taxanes (including 10-DAB III and paclitaxel). Although using absorbent as a dispersed agent showed more efficiency for cleaning up of a plant matrix, recovery percentage was not satisfactory. et al. This column was eluted using an aqueous-organic solvent including a water/methanol mixture. Percentages of methonal were optimized and the amount of packing for washing the most important polar and nonpolar taxanes such as paclitaxel and 10-DAB III. The fraction was analyzed using reversed-phase chromatography including C18 column. At the next stage fraction containing taxanes was loaded on to a homemade column, where a hydrophilic interaction solid phase extraction including a silica column was utilized according to the method cited in Hajnos ® HP-20 column followed by hydrophilic interaction SPE for obtaining polar and non-polar taxanes (paclitaxel and 10-DAB III). Final purification was done using semi-preparative reversed-phase liquid chromatography. + ion of paclitaxel and the observed mass at m/z of 554 corresponds to the [M+H]+ ion of 10-DAB III. To confirm separated compounds, liquid chromatography-mass spectrometry . Comparison of mass spectrometry data and previously reported data verified the structures of these separated compounds. In summary and as a conclusion, a combination of non-polar absorbents, hydrophilic interaction liquid chromatography solid phase extraction and reversed-phase chromatography demonstrates the ability for simultaneous separation and isolation of polar and non-polar taxanes. Also, as the solvent used in hydrophilic interaction liquid chromatography is compatible with reversed-phase chromatography, on line two-dimensional combinations could be used as a fast separation technique for isolation of natural products


Although deregulation of miR-146a has been reported in type 2 diabetes repeatedly, the direction of deregulation events (up or down) remained to be inconsistent in literatures. Therefore, in this study we performed a meta-analysis on the possible association between miR-146a expression levels and type 2 diabetes. A systematic literature searching of PubMed, ISI Web of Science and Google Scholar was performed up to the end of September 2016. Finally, a total of 12 studies including 344 diabetic patients and 316 controls were selected for meta-analysis. All statistical analysis was performed using the metafor package with R software. Moreover, publication bias was assessed by Egger’s and sensitivity analysis was applied on the meta-analysis. The results are presented as log10 odds ratios (logORs), 95% confidence intervals (CI) with relevant P values. The results revealed that miR-146a was downregulated in type 2 diabetes cases compared with normal subjects . Furthermore, sub-group analysis showed that the association between miR-146a expression levels and type 2 diabetes in whole blood (P<0.001) and PBMCs (P<0.001) samples were significant. However, this association was not significant in the serum (P=0.67) and plasma (P=0.90) samples. Our finding suggests that miR-146a downregulation could be associated with type 2 diabetes susceptibility. Further investigations with larger sample size are required to evaluate this association in the type 2 diabetes pathogenesis. Type 2 diabetes (T2D) is the most common form of diabetes resulting from interactionof genetic and environmental factors . This meSince the discovery in 1993, there has been a steady increase in the number of studies investigating the role of circulatory microRNAs (miRNAs) as highly stable and non-invasive biomarkers , 7. MiRNTo date, several studies have provided important evidence about the role of circulating miRNAs as biomarkers for T2D -14. The Search strategyA Systematic literature search using PubMed, ISI Web of Science and Google Scholar were performed for relevant articles from 1993 to September 2016 and the language was restricted to English. To find eligible articles, a combination of the following key words was used: “miR-146a”, “expression”, “profiling”, “type 2 diabetes”, “circulating”, “plasma”, “serum”, and “blood”. In additon, to identify additional eligible studies, we scanned the reference lists of reviews and publications selected for inclusion in this paper.Selection criteria and publication quality assessmentStudies were eligible if they met the following criteria: (1) had miRNAs expression profiling design, (2) compared patients with diabetes and healthy controls, (3) with information on cut-off criteria  for differentially expressed genes, (4) studied human circulatory miRNAs , and (5) reported total number of samples in both patient and control groups. We excluded review articles and studies that determined miRNAs expression profiling on human tissues.Data extraction and quality assessmentThe required data were retrieved by two independent investigators from the full- text eligible articles and supplementary materials. Any disagreement was resolved by a team discussion. For each study, the following information and characteristics were collected and recorded: the first author’s name, year of publication, paper DOI number, study population, miRNAs expression profiling technique, number of cases and controls, number of studied miRNAs, type of sample, cut-off value and type of dysregulation (upregulation or downregulation). With regardto the technique used in a given study, microarray platform or qPCR, quality assessment of data was performed according to the Minimum Information About a Microarray Experiment (MIAME) guidelines version 2 and Minimum Information for Publication of Quantitative Real-time PCR Experiments (MIQE).Statistical analysis2 statistics, respectively. If a difference in statistical heterogeneity was detected (PQ<0.10 or I2≥50%), a random-effects model (fitted by restricted maximum-likelihood estimator) was applied to the pooled logORs. Otherwise, the ﬁxed-effects model was used. To assess the stability of acquired P-values, leave-one-out  was applied on the meta-analysis. Additionally, since the sample sources  were different, a sub-group analysis was performed. Also, publication bias was examined by a funnel plot and funnel plot asymmetry was assessed by Egger’s linear regression test. The significance of the intercept was determined by the t test, as proposed by Egger, with P<0.10 considered indicative of statistically significant publication bias. Otherwise, the study was considered to have no publication bias.All analyses were performed using the metafor package with R statistical software 3.1. Meta-analysis was performed as described elsewhere . To asseThe characteristics of included studiesAfter titles and abstracts initial screening, 10 related articles were obtained by literature search from PubMed, ISI Web of Science and Google Scholar databases. Further, we added our work as unpublished study (consisted of two sub-studies). Finally, a total of 12 studies/sub-studies including 344 T2D patients and 316 normal samples were selected for meta-analysis .Meta-analysis of miR-146a expression in T2D2  was as 90.15%. Overall, meta-analysis revealed that miR-14a was downregulated in T2D cases compared with controls   . As a fu, -1.11) . Subsequ, -1.11) . Indeed,, -1.11)  and Rong, -1.11)  studies Finally, to evaluate the sample type effects on the meta-analysis, we performed sub-group analysis . Our anaT2D is a silent slow progressive killer disease resulting from chronic metabolic disorder in the body which is characterized by hyperglycemia, insulin resistance and finally pancreatic β-cell dysfunction . There iIRAK1)/TNF receptor-associated factor 6 (TRAF6), thus attenuating the inflammatory cytokines production in macrophages (Our results revealed that miR-146a was downregulated in T2D samples compared with controls. The miR-146a is one of the most important miRNAs that its deregulation has been implicated in diabetes. Accumulating evidence has reported that miR-146a regulates the genes involved in the pathogenesis of T2D and its related complications. It has been suggested that chronic inflammation is an important determinant in insulin resistance and also microvascular complications of T2D including nephropathy, neuropathy and retinopathy (rophages , 27. It rophages , 21. yetrophages , 14.Further, the subgroup analysis by sample type showed that the association between miR-146a expression level and T2D was significant in PBMCs and whole blood samples, whereas it was not significant for serum and plasma samples. As a result, determination of miR-146a expression level as molecular marker for T2D can be more useful in PBMCs than in serum and plasma. One possible reason for this finding may be due to the fact that miRNAs expressions are cell and tissue specific . It has Importantly, there is a report which performed a meta-analysis on miR-146a expression in different tissues in human and model organisms . AlthougEven though we tried to perform a well-designed and robust meta-analysis, the present study may suffer from some limitations. First, the existing bias in publishing of reports has significant result. Second, in the subgroup analysis using sample types, the number of studies in each group was relatively small. Third, methodological limitation in miRNAs expression meta-analysis analyzed all studies with non-significant results. Four, because our meta-analysis did not estimate the contribution of other risk factors related to T2D and also the stage of disease, these variables may have influenced the result of our study and partially explain the discrepancies in the involved case-control studies.In conclusion, our finding suggest that miR-146a downregulation could be associated with T2D susceptibility. Furthermore, this result suggests that miR-146a can be considered as a potent marker to predict the clinical outcome of diabetes. This association needs to be confirmed through more clinical investigations with larger sample size and well characterized study populations.


Staphylococcus epidermidis is a harmless human skin colonizer responsible for ~20% of orthopedic device-related infections due to its capability to form biofilm. Nowadays there is an interest in the development of anti-biofilm molecules. Marine bacteria represent a still underexploited source of biodiversity able to synthesize a broad range of bioactive compounds, including anti-biofilm molecules. Previous results have demonstrated that the culture supernatant of Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 impairs the formation of S. epidermidis biofilm. Further, evidence supports the hydrophobic nature of the active molecule, which has been suggested to act as a signal molecule. In this paper we describe an efficient activity-guided purification protocol which allowed us to purify this anti-biofilm molecule and structurally characterize it by NMR and mass spectrometry analyses. Our results demonstrate that the anti-biofilm molecule is pentadecanal, a long-chain fatty aldehyde, whose anti-S. epidermidis biofilm activity has been assessed using both static and dynamic biofilm assays. The specificity of its action on S. epidermidis biofilm has been demonstrated by testing chemical analogs of pentadecanal differing either in the length of the aliphatic chain or in their functional group properties. Further, indications of the mode of action of pentadecanal have been collected by studying the bioluminescence of a Vibrio harveyi reporter strain for the detection of autoinducer AI-2 like activities. The data collected suggest that pentadecanal acts as an AI-2 signal. Moreover, the aldehyde metabolic role and synthesis in the Antarctic source strain has been investigated. To the best of our knowledge, this is the first report on the identification of an anti-biofilm molecule form from cold-adapted bacteria and on the action of a long-chain fatty aldehyde acting as an anti-biofilm molecule against S. epidermidis. Staphylococcus epidermidis (S. epidermidis) is now being recognized as an important opportunistic pathogen that can cause significant problems when breaching the epithelial barrier, especially during the biofilm-associated infection of indwelling medical devices  and/or their complications , when grown with a sessile life-style, proved to be able to secrete an anti-biofilm molecule capable of inhibiting S. epidermidis biofilm formation ; P. haloplanktis TAC125  (ATCC®BAA-1117™) was grown as suggested by suppliers, cultivated in the Autoinducer Bioassay (AB) Medium 600nm; 100 μl of opportune dilutions of pentadecanal in AB medium were added into each well. Luminescence and (OD)600nm were monitored every 15 min over 18 h with a high-performance multimode plate reader  in order to correlate the effects of the products on both the growth and bioluminescence kinetics. Data are reported as light units (LU).To screen the ability of the compounds to interfere with QS, the test products were serially diluted in the AB medium using sterile, black, clear-bottom 96-well microtiter plates (Greiner Bio-one #655090) as assay platform. An overnight culture of P. haloplanktis TAC125 bacterial culture was grown in BHI medium in a Stirred Tank Reactor 3 L fermenter (Applikon) connected to an ADI-1030 Bio Controller (Applikon) with a working volume of 1 L. The bioreactor was equipped with the standard pH-, pO2-, level-, and temperature sensors for the bioprocess monitoring. To allow the biofilm formation, autoclaved solid polystyrene supports were added into the bioreactor (33 supports in 1 L). The culture was carried out at 15°C for 48 h, or at 4°C for 96 h, in aerobic conditions using airflow of 6 L/h, without stirring. After incubation, supports were removed from the supernatant and the adherent cells were recovered by sonication as previously described  equipped with control units for pH and temperature, an oxygen sensing electrode measuring the solution oxygen pressure, and mechanical stirring (200 rpm) at 4°C. Each fermentation unit, filled with medium and sterilized by autoclaving, was equilibrated at process temperature. In order to allow oxygenation of the medium, the stirring speed was set up at the maximum value to be used during the fermentation, and then sterile air supply was switched on. The system was left to stabilize for at least 30 min to guarantee the saturation of the medium with air. In these conditions 100% of measured oxygen pressure was assigned. The zero-point set calibration was performed by saturating the medium with sterile nitrogen gas. Under aerobiosis conditions, measured oxygen pressure was maintained always above 20% by modifying stirring speed and aeration rate. In microaerobiosis  air supply was stopped after inoculum. For each strain, the growth kinetics were followed in triplicate in at least two independent experiments.Standard methods were employed for DNA manipulation and isolation, amplification by PCR, and DNA sequencing. Restriction enzymes, T4 DNA ligase, alkaline phosphatase, T4 polynucleotide kinase, Klenow fragment, Taq DNA polymerase were supplied from Boehringer-Roche, Amersham-Pharmacia Biotech, Promega, and New England Biolabs. DNA fragment purification was carried out with the QUIAEX II kit from Qiagen GmbH.P. haloplanktis TAC125 genomic DNA was used as PCR template and two primers were designed to amplify a 275 bp-long region of the PHSAb0219 gene and to introduce an EcoRI and a SphI site (Oligo b0219EcoRIfw 5′-CTATGAATTCAAGAAGATATTTACGAGC-3′ and Oligo b0219SphIrv 5′-AATACCCGCATGCCGTTGGTGCC-3′). The amplified DNA fragment was digested by EcoRI and SphI, and inserted into the pVS plasmid  as selection agent.PCR was employed to amplify a DNA fragment of PSHAb0219 gene. P. haloplanktis TAC125 cells recovered from both planktonic growth and, after sonication of supports, biofilm growth as reported in literature  according to the manufacturer's instructions. The reaction mix was amplified . For each reverse transcriptase amplification, an additional PCR reaction on DNA-free total RNA was performed as control, in order to exclude false positive signals  glacial acetic acid and 80% (v/v) ethanol. After 30 min of incubation at room temperature, (OD) was measured at 590 nm to quantify the total biomass of biofilm formed in each well. Each data point is composed of three independent experiments, each performed at least in 3-replicates.2 for 2 min. Coating with 100 μl of 10 μg/ml fibronectin was made for 2 min at 1 dyne/cm2. The fibronectin binding was performed for 30 min without flow. After priming, fibronectin was aspirated from the output wells and replaced with 100 μl of fresh overnight cultures diluted to an (OD)600 of 0.8. The channels were seeded by pumping from the output wells to the input wells at 2.0 dyne/cm2 for 4 sec. Bacterial adhesion was performed for 30 min at 37°C without flow. 2.0 mL of BHI was added to the input well and pumped at 1 dyne/cm2 for 12 h. We used two inlet wells; in the first well we added only BHI. In the second well we added pentadecanal at a concentration of 120 μg/ml. Bright-field images were taken at 40X magnification at 1-min intervals for a total of 720 time points.To continuously monitor the biofilm development in dynamic condition, we utilized a BioFlux 2,000 microfluidic system , which allows the acquisition of microscopic images over time using the experimental protocol previously set up  was then purified on a gel-filtration column  eluted with MilliQ water. The active fractions were collected and fractionated on a C18 reverse phase column , eluted with CH3CN:H2O ranging from 10 to 95% of CH3CN. The fraction resulted to be active (S) was analyzed on a Agilent Technologies gas chromatograph 6850A equipped with a mass selective detector 5973N and a Zebron ZB-5 capillary column , by using the following temperature program: 150°C for 3 min, from 150 to 300°C at 15°C/min, at 300°C for 5 min.As reported in the previous paper . 1- heptadecanol  was charged into a 10 mL round-bottom flask equipped with a magnetic stir bar. The solid was then dissolved in toluene (2 mL) at 20°C, and an aqueous solution of sodium bicarbonate  was prepared and charged into the toluene slurry. Solid iodine  was then charged to the alcohol followed by solid TEMPO 600nm. The bacterial cultures were incubated at 37°C for 20 h in presence of pentadecanal (100 μg/ml) in order to assess its anti-biofilm activity and its influence on cell viability.For confocal microscopy biofilms were formed on Nunc™ Lab-Tek® 8-well Chamber Slides . Briefly, overnight cultures of The biofilm cell viability was determined with the FilmTracer™ LIVE/DEAD® Biofilm Viability Kit  following the manufacturer's instructions. After rinsing with filter-sterilized PBS, each well of the chamber slide were filled with 300 μl of working solution of fluorescent stains, containing the SYTO® 9 green fluorescent nucleic acid stain (10 μM) and Propidium iodide, the red-fluorescent nucleic acid stain (60 μM), and incubated for 20–30 min at room temperature, protected from light. All excess stain was removed by rinsing gently with filter-sterilized PBS.All microscopic observations and image acquisitions were performed with a confocal laser scanning microscope  equipped with an Ar laser (488 nm), and a He-Ne laser (555 nm). Images were obtained using a 20X/0.8 objective. The excitation/emission maxima for these dyes are ~480/500 nm for SYTO® 9 stain and 490/635 nm for propidium iodide. Z-stacks were obtained by driving the microscope to a point just out of focus on both the top and bottom of the biofilms. Images were recorded as a series of.tif files with a file-depth of 16 bits. For each condition, two independent biofilm samples were used.P. haloplanktis TAC125 inhibits S. epidermidis biofilm formation and that the bacterium produces the active molecule only when it is grown in a sessile condition . The obtained cell-free supernatant was recovered after 96 h and separated from the floating polystyrene supports and the cells by a centrifugation at 13,000 rpm. Subsequently the supernatant was sterilized by filtration through membranes with a pore diameter of 0.22 μm, and then underwent dialysis treatment using a semipermeable membrane with a cut-off of 3.5 KDa. The permeate product after lyophilization was fractionated on a Biogel P-2 column and the anti-biofilm activity of each fraction was evaluated (data not shown). The fraction endowed with anti-biofilm activity on S. epidermidis O-47 was further purified on a C18 reverse phase column eluted with acetonitrile/water. Several fractions (A–S) were collected, and the anti-biofilm activity of each fraction was tested as reported in Figure S. epidermidis biofilm. The analysis of the 1H NMR spectrum of this fraction revealed a signal at δ 9.77 ppm, which immediately suggested the presence of an aldehyde (MOLBASE.COM CAS. No. 2765-11-9). This was believed to be aliphatic, due to the presence of intense signals in the range between δ 0.1–2.5 ppm (data not shown). To confirm this hypothesis, and to check the purity of the sample, a GC-MS analysis was performed , 1H-1H TOCSY , 1H-13C DEPT-HSQC (distortionless enhancement by polarization transfer-heteronuclear single quantum coherence), and 1H-13C HMBC (heteronuclear multiple bond correlation) experiments were performed. The proton at δ 9.79 ppm, that clearly indicated an aldehyde functional group on the molecule, displayed a correlation with a carbon at δ 203.1 ppm  that constitute the aliphatic chain. The last signal, attributable to the CH2C14 (δH/C 1.36/22.9), showed a long-range cross-peak, with the methyl group at δH/C 0.9/13.6 ppm .Once the structure of the anti-biofilm molecule had been identified, the first step was to confirm the absence of any antimicrobial activity of pentadecanal on the staphylococcal reference strains by determining the minimum inhibitory concentration. The data obtained indicated no bacteriostatic and no bactericidal activity within the range of concentrations explored . Preliminary experiments were also carried out to assess the effects of the molecule on the planktonic growth rate of S. epidermidis strains . All previously described experiments were performed in BHI medium. The pentadecanal anti-biofilm activity was also tested in TSB medium to verify its effectiveness in function of growth medium composition. The results demonstrated that this latter did not influence the anti-biofilm ability of pentadecanal .The pentadecanal activity on biofilm formation was assessed on two different s Figure , accordiS. epidermidis O-47 at different times, in the absence or in the presence of pentadecanal . The bacteria were seeded in both channels visible in each frame and after 30 min the flow was applied. The images collected showed an initial rapid growth of the bacteria, resulting in a confluent “lawn” of cells that was followed by a period of detachment. Pentadecanal clearly impaired the biofilm formation confirming the results obtained in the static system.The pentadecanal anti-biofilm efficacy was also tested in dynamic conditions with the BioFlux system, a microfluidic device that precisely controls the flow of growth medium between two interconnected wells of a microtiter plate. By positioning the channel connecting the two wells over a window accessible for viewing by microscopy, biofilm growth can be monitored in a time-course assay in which images are collected at 1-min intervals. In Figure S. epidermidis O-47 biofilm was further investigated by confocal laser scanning microscopy  that are essential for the anti-biofilm activity, the effect of similar aldehydes and corresponding alcohols, characterized by different lengths of the aliphatic chain in the range from C-14 to C-20, were analyzed and the results are summarized in Figure V. harveyi BB170 strain (luxN::tn5Kan), a mutant sensor strain that responds only to AI-2 autoinducers. The V. harveyi BB170 bioluminescence was monitored  pentadecanal reduced the bioluminescence . did not allow us to identify the genes coding for enzymes known to be able to synthesize fatty aldehydes, like fatty acid reductase . The aldehyde oxidoreductase, in specific conditions, could be able to produce pentadecanal by reducing the corresponding fatty acid. To assess if this was a correct hypothesis, we first investigated the presence of the pentadecanoic acid in P. haloplanktis TAC125 cells. A GC-MS analysis of fatty acid methyl esters from P. haloplanktis TAC 125 cells, grown either in sessile or in planktonic conditions, revealed the presence of pentadecanoic acid . The results of the RT-PCR experiments  necessary for cell metabolism. To verify this hypothesis P. haloplanktis TAC125 cells were grown, in an automatic bioreactor, at 4°C in microaerobiosis under agitation, a condition where the measured oxygen pressure is kept constantly below 5% saturation (measured oxygen pressure <5%). The anti-biofilm activity of the cell-free supernatant of P. haloplanktis TAC125 grown in microaerobiosis (SN-M) was tested on S. epidermidis biofilm. The SN-M proved to be able to inhibit the S. epidermidis O-47 biofilm formation .Although pentadecanal could be not defined as a strictly biofilm-specific metabolite, its effect on t Figure  on the AStaphylococcus epidermidis as an opportunistic pathogen is closely related to the biofilm forming capability of this bacterial species, especially during the biofilm-associated infection of indwelling medical devices. The increasing use of implanted medical devices heightens the importance of S. epidermidis as a human pathogen. Our research has been aimed at discovering anti-biofilm molecules from natural sources, such as marine microbiota, since we are convinced that this approach will lead to the discovery of novel and unforeseen compounds. Cold-adapted marine bacteria represent a still underexploited source of biodiversity able to synthesize a broad range of bioactive compounds, including anti-biofilm molecules. Previous results . In contrast, pentadecanal lacks any antibacterial activity against free-living bacteria and against bacteria entrapped in biofilm matrix. Indeed as demonstrated by CLSM analysis the presence of pentadecanal reduces the biofilm thickness without affecting the viability of S. epidermidis biofilm has been demonstrated by testing chemical analogs differing either in the length of the aliphatic chain or in their functional group properties. The results reported demonstrate that both the length of the aliphatic chain and the functional group properties are essential for the pentadecanal activity against S. epidermidis biofilm.The specificity of pentadecanal action on S. epidermidis biofilm could be related to its possible interference in the quorum sensing system. The most important and best-characterized quorum-sensing system in staphylococci is the accessory gene regulator (agr) system. Remarkably, the P. haloplanktis TAC125 anti-biofilm molecule is active on the clinical isolate O-47, which is a naturally occurring agr mutant  negatively regulates the expression of the ica gene [ica operon encodes enzymes responsible for the production of polysaccharide intercellular adhesion PIA , key molecules able to lead to biofilm detachment of S. epidermidis biofilm in vivo e in vitro +-dependent catalyses, in oxygen limitation, the reduction of the pentadecaonic acid to aldehyde to obtain the oxidized cofactor (NAD(P)+) necessary for cell metabolism. Therefore, the pentadecanal production occurs in the biofilm condition and in microaerobiosis due to the reduced oxygen availability that characterizes these two growth conditions. The oxygen availability results a crucial parameter for pentadecanal synthesis and proves to be the main difference between sessile and planktonic conditions, so determining the anti-biofilm production. Moreover, the results described in this paper indicate that the long-chain fatty aldehyde is not involved in the control/modulation of the Antarctic bacterium biofilm development.Besides the characterization of pentadecanal anti-biofilm activity on S. epidermidis biofilm-associated infections.In conclusion, this is the first report both on the action of a long chain fatty aldehyde as an anti-biofilm molecule and on an aldehyde working as a signaling molecule in the AI-2 QS system. We believe that this paper endorses the potential of cold-adapted marine bacteria as a source of bioactive compounds of interest, and contributes to the development of innovative approaches for the prevention and treatment of AC: Performed the experiments, suggested critical parameters in design of experiments and co-wrote the paper. RP: Performed the experiments, suggested critical parameters in design of experiments and co-wrote the paper. AR: Performed the experiments and co-wrote the paper. FS, MZ and MT: Performed the experiments. LS: Provided advice in performance of experiments and edited paper. GM: Provided advice in performance of experiments and co-wrote paper. MC: Suggested critical parameters in design of experiments and co-wrote paper. MLT: Suggested critical parameters in design of experiments and co-wrote paper. MA: Provided advice in performance of experiments and edited paper. EP: Designed the experiments, provided advice in performance of experiments and wrote the paper.This work was supported by Programma Nazionale di Ricerche in Antartide 2013/B1.04 Tutino.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this work, glyceryl trimyristate (Dynasan 114) nanoparticles containing the drug LPM and sodium cholate as a stabilizer were prepared using a modified solvent evaporation technique. Two LPM-loaded SLNs, namely LPM-SLN-1 (LPM-SLN with a high ratio rate of lipid to drug) and LPM-SLN-2 (LPM-SLN with a low ratio rate of lipid to drug), were prepared by the solvent evaporation method. A change in the lipid concentration affects the characteristics of LPM-SLNs. The average sizes of the LPM-SLNs were 303 ± 18 nm and 519 ± 36 nm, separately, as analyzed by dynamic light scattering. The LPM-SLNs were found to be round with a smooth surface, as observed using a transmission electron microscope and a scanning electron microscope. The average encapsulation efficiencies were 87 ± 3.78% w/w and 84 ± 5.17%, accordingly. In the in vitro release experiments, LPM-SLNs showed a continuous release profile of LPM without any burst release. The oral bioavailability of LPM-SLNs was analyzed using Wistar rats. The relative bioavailabilities of LPM-SLNs were 227 and 153%, respectively, as compared that of the LPM tablet. There was no difference in the Tmax between LPM-SLN-2 and the LPM tablet. In conclusion, LPM-SLN-1 significantly improved the oral bioavailability of LPM, while LPM-SLN-2 having the same swift action as the LPM tablet. These results demonstrate the potential of LPM-SLNs in the oral delivery of LPM to treat different types of diarrhea.The aim of the project was to assemble two optimum solid lipid nanoparticle (SLN) formulations for oral delivery of loperamide (LPM) to treat different types of diarrhea, and to evaluate their release profiles  L-lactide) nanoparticles to increase its solubility and to improve its release profile in vitro (Loperamide (LPM) is an orally administered antidiarrheal agent indicated for the short-term treatment of acute and chronic diarrhea in adults . LPM is in vitro . HoweverDelivering drugs to their site of therapeutic action is one of the main challenges faced by the pharmaceutical industry . This caOne important area of nanoencapsulation is the development SLNs. SLNs encompass a broad range of colloidal systems, which can include micro emulsions, liposomes, and polymeric nanoparticles . The nanin vitro, and its oral bioavailability was analyzed in vivo.The aim for the study is to assemble stable LPM-loaded SLNs and further explore the possibility of using these novel formulations to cure different kinds of diarrhea. LPM-loaded Dynasan 114 SLNs were prepared using the solvent evaporation method, with modifications . The forChemicals used for the synthesis of nanoparticles were loperamide , sodium cholate hydrate , glyceryl trimyristate , and dichloromethane . Purified water  was used throughout and all other chemicals were of analytical grade. All experiments were conducted at room temperature (25°C). Loperamide tablets were purchased from Xian Janssen Pharmaceutical Ltd, Xi’an, China.Table 1 and referenced by identification name.The loperamide-loaded SLNs (LPM-SLNs) were prepared by a solvent evaporation technique, with modifications . The nonThe particle size and PDI were determined by dynamic light scattering . Particle size, PDI, and ZP measurements of the SLN dispersion were performed immediately after manufacturing, and again after the dispersion had been freeze dried and reconstituted in purified water. In all cases, the dispersion was measured immediately after it was allowed to equilibrate for 2 min in the equipment.The morphology of LPM-SLN formulations was studied using transmission electron microscopy . The injection volume was 20 μl; a mixture of 0.1 mol/l ammonium carbonate and acetonitrile  was used as a mobile phase with a flow rate of 1.0 ml/min. The UV detection wavelength was 264 nm; the column temperature was 30°C.The mass analysis of LPM was performed by reverse-phase HPLC. The HPLC system consisted of a model LC-10AT pump  and a model SPD-10A UV detector . The frozen samples were then freeze dried  over 48 h to yield a powdery sample. To determine whether the freeze-dried powder could be reconstituted into SLNs, 30 mg of the powder was resuspended in 3 ml of purified water to yield the same concentration of SLNs (1% w/v) as was present in the initial dispersion prior to freeze drying. To aid the dispersion of the SLN, the resuspended sample was either vortexed for 5 min at 2400 rpm  or sonicated for 2 min at 80% amplitude (Vibra cell Model VCX130).The release rates of LPM from LPM-SLN-1, LPM-SLN-2, and LPM tablets were investigated using dialysis bags at pH 1.2 and pH 6.8. LPM-SLN-1, LPM-SLN-2, and LPM tablets equivalent to 10 mg of LPM were placed into a dialysis bag  containing 1000 ml PBS (pH 1.2 or 6.8) with 1% Tween 80, at 37.5°C. At different time points, the release medium in which the dialysis bag was immersed was sampled (5 ml) for analysis by the above mentioned HPLC method.All animal experiments were carried out following the Principles of Laboratory Animal Care  or an equivalent dose of the LPM tablet by gavage at a dose of 5 μl/g of body weight. Blood samples were collected as previously reported . BrieflyThe concentrations of LPM in rat plasma were determined by HPLC as mentioned above. Samples were prepared as following: 0.4 ml plasma was mixed with 0.2 ml 0.3 M NaOH and vigorously vortexed for 1 min. Then, 2 ml of ethyl acetate was added and the mixture was vortexed for 5 min. The organic layers were collected after centrifugation at 5000 rpm for 5 min, and evaporated to dryness. The residue was dissolved in 0.2 ml of the mobile phase by vortexing for 5 min and 20 μl was injected into the HPLC.Cmax) and time of maximum concentration (Tmax) were read directly from the concentration–time curves. Plasma concentration vs. time data were fitted into a two-compartment model to obtain estimates of the area under the plasma concentration–time curve during the period of observation (AUC0-t). The relative bioavailability of the SLN formulations was calculated by the following formula:The major pharmacokinetic parameters were determined by DAS 2.0 . The values of maximum concentration , R was the reference formulation . The differences were considered significant when p < 0.05.All batches were produced in triplicates except when mentioned otherwise. All values are expressed as mean ± SD. Statistical analysis of the mean values between batches was performed using the Student’s Figure 1A). ZP measurements suggested that LPM-SLN-1 possessed a high negative surface charge of -47.33 ± 4.29 mV.The DLS measurements for LPM-SLN-1 showed a mean size of 303 ± 18 nm, with a PDI of 0.19 ± 0.01. A histogram of the size distribution showed a peak at 342 nm and a normal distribution with size range of 68-1484 nm . The particle sizes of three batches of LPM-SLN-1 and LPM-SLN-2 were 296.65 nm, 323.75 nm, and 289.6 nm; and 526.84 nm, 550.69 nm, and 479.47 nm, respectively. The MD of the two formulations was significantly different (p = 0.00295). However, LPM-SLN-2 did not show a statistically significant difference in ZP (p > 0.05) when compared to LPM-SLN-1. Interestingly, there was no increase in the PDI (p > 0.05), confirming that an increase in the drug load in LPM-SLN-2 did not widen the nanoparticle size distribution, as compared to LPM-SLN-1.The DLS measurements for LPM-SLN-2 showed a mean size of 519 ± 36 nm, with a PDI of 0.18 ± 0.01 and ZP of -54.63 ± 8.12 mV. The size distribution histogram showed a slightly broader continuous distribution with a peak at 458 nm and a slight positive skew between 122 and 1484 nm . The sonicated samples had similar MD and PDI values as the freshly prepared samples. These experiments suggest that freeze-dried LPM-SLNs were fairly well reconstituted by sonication. Lyophilization is a possible way to preserve LPM-SLNs.The LPM-SLN samples were freeze dried to determine whether the SLNs were amendable to freeze drying. The freeze-dried samples were reconstituted in purified water by either vortexing or sonication. As shown in Figure 4. The release profiles of LPM-SLNs at pH 1.2 and pH 6.8 showed no burst releases of LPM at the beginning in both media, which verifies that there was little drug attached to the surface of SLNs. A continuous release profile was observed at pH 6.8. The accumulated dissolution of LPM from SLNs in PBS (pH 6.8) over 48 h was about 80%, whereas, it was less than 20% from commercial tablets (The LPM release profiles at different pHs are shown in  tablets . HoweverR = 0.8071 ln t- 0.2452 (r = 0.9896) for LPM-SLN-1 and ln R = 0.7549 ln t - 0.3147 (r = 0.9934) for LPM-SLN-2. Based on the fitting result using the Ritger–Peppas model , which indicated that LPM release from SLNs was due to drug diffusion and lipid matrix corrosion.  were fitted to a Ritger–Peppas kinetics model using Origin 8.5 and the following equations were obtained: ln rrosion.  The k vaFigure 5 shows the plasma concentration-time profiles of LPM after oral administration of different LPM formulations to male Wistar rats. The corresponding PK parameters are given in Table 2. The Tmax was 0.33 h and the Cmax was 461.72 ± 49.15 ng/ml when the LPM tablet was orally administrated. The Tmax of LPM-SLN-1 (2 h) was about one and half hour later than that of the LPM tablet. The difference between Tmax values of LPM-SLN-1 and the LPM tablet demonstrated that the absorption modes of the two formulations were different. The LPM in the tablet dissolved in the intestinal tract and was absorbed directly into the systemic circulation. Therefore, the plasma concentration of LPM quickly reached the plateau in 0.33 h. However, the drug from LPM-SLN-1 was slowly released into the gastrointestinal tract, which was verified by the in vitro release experiments. Interestingly, the Tmax values were not significantly different between LPM tablet and LPM-SLN-2, which may be attributed to the bigger size of LPM-SLN-2, leading to an easy diffusion of the drug from the lipid matrix. The Cmax values of LPM-SLN-1 and 2 were 731.87 ± 43.89 ng/ml and 638.98 ± 50.06 ng/ml, respectively, which were substantially higher than that of the LPM tablet (461.72 ± 49.15 ng/ml). At all except the first three time points, the LPM plasma concentrations in rats treated with LPM-SLNs were significantly higher than those treated with the LPM tablet. . tablet.  Sixteen in vivo experiment, relative bioavailability of SLN formulations is determined by dividing AUC0-t of LPM SLN formulations by AUC0-t of LPM tablet. Thus, Frs of LPM-SLN-1 and LPM-SLN-2 were 227 and 152%, correspondingly, which meaned more LPM in SLNs crossed intestinal barrier into systemic circle, compared to LPM tablet.Since the equal amount of LPM in SLN formulations and tablet was used for Cmax and sustained release profile of LPM-SLN-1 would result in better treatment of chronic diarrhea. While LPM-SLN-2 had similar Tmax as the LPM tablet, its profile makes it more suitable for treatment of acute diarrhea. Overall, the LPM-SLNs indicate an encouraging potential of increasing the oral bioavailability of water-insoluble drugs along with an ability to mask unpleasant taste.The results suggest that the systemic absorption of LPM was drastically improved by incorporation into SLNs as compared to tablets . Higher The main factors contributing to the low bioavailability of LPM are its poor water-solubility, significant first pass effect in the liver, and the obstacle of the intestinal epithelial cell. Hence, we used SLNs to overcome these problems and propose the mechanism by which the effects are obtained.First of all, SLNs are composed of different lipids whose structures are similar to the fat found in foods. The lipids could stimulate bile secretion to the small intestinal and the LPM released from SLNs was mixed with bile salts to form micelles which would improve its solubility .in vivo.Secondly, as LPM is a weakly alkaline molecule, it forms salt at a low pH (1.2), which subsequently increases its solubility. After oral administration of LPM tablets, the drug would quickly dissolve in the gastric fluid ; and upop-glycoprotein irreversibly and inhibit the p-glycoprotein eﬄux pump, which would increase the attachment of LPM to the intestinal mucosa  were initially introduced . In thesWassim The water solubility of loperamide is very limited (about 0.00086 mg/mL at 20°C). Tween 80  is a hydrophilic non-ionic surfactant widely used in emulsification and dispersion of substances in medicinal and food products . Hence, 50 of LPM for oral administration in rats is 185 mg/kg. Oral doses (2.5–40 mg/kg) several times the antidiarrheal dose, unlike diphenoxylate, do not produce narcotic-like actions (in vivo experiments.LD actions . In our In vitro experiments revealed a sustained release of the drug by LPM-SLNs, with little burst-release effect at pH 6.8. In vivo pharmacokinetic experiments indicated that the relative bioavailabilities of the LPM-SLN formulations were 227 and 153% higher than that of the LPM tablet in Wistar rats after oral administration. The SLNs could increase the gastrointestinal absorption of LPM. The nanostructured lipid vehicle provides a promising way to improve the oral bioavailability of water-insoluble drugs. The two different formulae were designed for different clinical usages. Based on these results, further investigations would be carried out to establish the exact mechanism underlying increased LPM oral absorption by this formulation.In this project, LPM-SLNs for oral delivery were successfully formulated by a solvent evaporation technique. The resultant LPM-SLNs formulations had relevant and homogeneous particle size with high encapsulation efficiency. It could be preserved and easily restored after lyophilization. LW and YY set up and carried out experiments, KS set up HPLC method and analyzed samples, JW executed data analysis, JM and WZ lead the research.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Classical swine fever (CSF) is a major pig disease worldwide . Some rey-axis), it is clear that the Ne for CSFV-G1 was higher (around 102–2.5) compared to the Ne values of CSFV-G2 until the year 2000. Between 2000 and 2005, there is a sudden increase in the Ne for CSFV-G2 followed by an almost equal decrease. The Ne values remain higher for CSFV-G1 compared to CSFV-G2, plateauing at approximately 102.5 after 2009 at the time when the genetic diversity of the CSFV-2 continues to decrease. Moreover, any comparison after this point is difficult to assess since the authors did not continue their analysis for CSFV-G1 after 2010, unlike that of CSFV-G2 (Analyzing the global evolutionary patterns for CSFV, Yoo et al.  stated taround 10–2.5 comp CSFV-G2 –5. FinalCSFV-G2 has a more advantageous E2 codon composition than CSFV-G1, in terms of survival in immune environments that have been optimally created by CSFV genotype 1-based vaccination.” This conclusion was not supported by the methods used. For the evaluation of the selective pressure on CSFV, Yoo et al. (Second, the author’s state: “o et al. . For thio et al.  showed, o et al. . When uso et al. .Because of the emergence of neutralization-escape mutants from the CSFV-G2 strains caused by the disproportionate use of MLV based on CSFV-G1, Yoo et al. (A series of studies , 7–9 preRelevant factors that could facilitate the emergence neutralization-escape mutants for CSFV were omitted in Yoo et al. . These iLR and LP wrote the manuscript. LP edited the manuscript. Both authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


R2 and area under receiver operating characteristic curve [AUC]) and two bootstrap procedures were used for internal validation. Factors associated with the probability of SL TT eligibility were the presence of brain metastases , number of metastatic sites , platelet count , lactate dehydrogenase level , and albumin concentration . We developed on-line calculator that enables practicing clinicians to estimate SL treatment probability .In order to facilitate long-term treatment decisions, we aimed to define biomarkers defining the probability of receiving second-line (SL) targeted therapy (TT) in patients with metastatic renal cell carcinoma (mRCC) based on their characteristics present at first-line TT initiation. We analysed 152 consecutive mRCC patients treated and used multivariable binominal logistic regression to identify factors contributing to the probability of receiving SL TT. Final model was assessed with bias-corrected indices (Nagelkerke’s  Currently multiple antiangiogenic compounds including bevacizumab , sorafenib, sunitinib and pazopanib (tyrosine kinase inhibitors [TKIs] targeting VEGF receptors) are first-line standard-of-care treatment options of metastatic renal cell carcinoma (mRCC) providing progression-free survival (PFS) benefit as proven in randomised phase III trials . NeverthNational cancer treatment programmes and/or drug prescription registries that cover the whole country populations provided data on new anti-mRCC therapies within real-life patients , 9. CurrUntil now, the Memorial Sloan-Kettering Cancer Center (MSKCC) classification score (Motzer Score) and first-line treatment type were considered as only established predictive factors of receiving second-line therapy . MoreoveConsecutive mRCC patients who started treatment with first-line TT between November 2009 and March 2016, in the Department of Oncology, Military Institute of Medicine in Warsaw, Poland were included in the analysis. Patients with any histological RCC subtype with no other primary malignancies and no adjuvant therapy were eligible. Additionally, patients who were treated with interferon-based immunotherapy prior to the initiation of first-line TT were included; however, IFN was not counted as a line of treatment. Patients were assigned to the second-line (SL) group if they had received any of the second-line TT therapy, or to the non-SL group, if they had not received any therapy beyond first-line. Patients with unknown status of second-line TT were excluded from analysis. This group comprised of patients who (1) continued treatment as had not progressed on first-line TT at the time of the final data collection or (2) discontinued treatment due to toxicity/consent withdrawn, not progression or (3) were lost to follow-up before second-line TT initiation. Inclusion criteria for FL and SL covered adequate organ function as described before –19.The individual medical records were analysed. The institutional ethics committee approved the study (agreement no. 48/WIM/2014). Due to retrospective design of the analysis, individual informed consent was not required.The status of second-line TT (received versus not received) was a dependent binary variable for the main analysis. The other assessed outcomes were (1) OS which was defined as the time from the initiation of first-line TT to death from any cause, (2) PFS which was defined as the time from the initiation of first-line TT to disease progression according to the Response Evaluation Criteria in Solid Tumours (RECIST), version 1.1, or death from any cause, and (3) post-progression survival (PPS), which was defined as the time from disease progression on first-line TT to death from any cause. Medians and ranges were used to describe continuous variables whereas frequencies and percentages were used to describe categorical variables. The differences in baseline characteristics between the SL and non-SL groups were assessed using the U-Mann-Whitney test for continuous variables and the Pearson Chi-Square or the Fisher’s exact test (in the case of five or less expected frequencies in each cell of a studied contingency table) for categorical variables. Distributions of OS, PFS and PPS were estimated using the Kaplan–Meier product-limit method; their medians with calculation of 95% confidence interval (CI) using log–log transformation were reported. The differences in survival probabilities between the SL and non-SL groups were assessed using the log-rank test. The median follow-up time was calculated using the Schemper and Smith method . PatientP value less than 0.1 were included in the second step, i.e. multivariable analysis based on step-wise forward selection with significance level of 0.05 for entering and removing variables. Factors that remained significant in the second step contributed to the final model. The model performance was assessed with Nagelkerke’s R2 and bias-corrected Nagelkerke’s R2 as global goodness-of-fit measures, the Hosmer–Lemeshow test for calibration, an area under receiver operating characteristic curve (AUC) and bias-corrected AUC for discrimination.The identification of factors that independently predicted receiving second-line therapy was conducted using a two-step procedure based on binominal logistic regression. In the first step, all factors were included in univariable analysis and these factors that reached P values were produced and compared to those of the model derived from original dataset. In the second procedure, another 1000 bootstrap datasets entered the same modelling process used to derive the final model from the original dataset. Factors that appeared in more than 50% of computed models were considered to be significant ) patients had known second-line TT status and, therefore, were included in the analysis. One hundred and fifty-two (152 [57%]) patients had received second-line TT  and contributed to the SL group. The remaining 115 [43%] patients were not eligible to receive any subsequent systemic treatment and were assigned to the non-SL group. The detailed characteristics collected at the time of first-line TT initiation are presented in Table 00%) patiP = 0.496). The median PFS was 8.0 months (95% CI 6.7–9.4), the median OS was 20.0 months (95% CI 17.5–24.7) and the median PPS was 7.7 months (95% CI 6.1–10.2) for all analysed patients. The median PFS was 11.7 months (95% CI 9.0–14.1) and 4.9 months (95% CI 3.5–5.6) for the SL-group and the non-SL group, respectively. The median OS was 30.4 months (95% CI 26.2–37.8) and 7.4 months (95% CI 5.5–10.3) for the SL-group and the non-SL group, respectively. The median PPS was 14.9 months (95% CI 13.5–16.7) and 1.9 months (95% CI 1.2–3.0) for the SL-group and the non-SL group, respectively. The SL-group had significantly longer PFS (P < 0.001), OS (P < 0.001) and PPS (P < 0.001) than the non-SL group . The median follow-up time was 69.2 months (95% CI 58.5–74.7) in the SL-group and 71.2 months (95% CI 65.0–78.0) in the non-SL group, respectively. The follow-up time did not differ between the two groups (4/ml) and lactate dehydrogenase (LDH) level , while albumin concentration was associated with increased probability . The model showed satisfactory calibration , discrimination  and global fit . In the first validation procedure, all five model covariates remained statistically significant after repeating the regression on 1000 bootstrap samples. In the second validation procedure, four factors: number of metastatic sites, LDH, platelet count and albumin concentration appeared in more than a half of 1000 newly constructed models , whereas the brain metastases status did not (45%)  in the second bootstrap procedure remained significant. The brain metastases status did not reach the planned 50% frequency of entry probably due to statistical uncertainty caused by small proportion of patients who had brain metastases .The median follow-up time in our study was about was almost six years which is one of the longest reported in the literature , 25. SucNowadays, everolimus, axitinib, nivolumab and cabozantinib are used extensively in patients who progressed on prior antiangiogenic TKI therapy. Currently with multiple treatment options, including immunotherapy, reimbursed in selected countries, optimal choice and sequencing is more and more challenging . The BayThe limitations of the study include its retrospective design and lack of external validation in another cancer centre. Nevertheless, the proposed model was successfully validated using two internal bootstrap procedures and has shown good statistical performance. Similar models, including nomograms and calculators are being developed in the field of medical oncology practice including advanced oesophagogastric adenocarcinoma nomogram for patients undergoing first-line combination chemotherapy , advanceThe MRCCSECLINE calculator developed in our study may be a useful tool for clinicians to identify those mRCC patients, who are unlikely to receive second-line treatment, and subsequently, to help determine the most optimal, long-term treatment plan at the beginning of systemic TT. However, independent validation of the calculator in prospective trials and additional studies to identify other tumour-specific prognostic factors for all therapies are needed in the future. Medical calculator potentially facilitates evidence-based treatment decisions, individualised risk assessment and helps to select suitable agents or BSC for second-line treatment of mRCC.Until today, the MSKCC score and first-line treatment type were considered as predictive factors of receiving second-line therapy. No second-line treatment-oriented nomograms or prediction scales are available. We have evaluated 17 clinical and biochemical parameters that are widely evaluated at RCC first line treatment initiation and defined these that impact first-line treatment survival and, therefore, second line treatment enrolment. The presence of brain metastases, number of metastatic sites abnormal platelet count and lactate dehydrogenase level are found in patents that are at a risk of nor eligibility of second line treatment. Normal albumin concentration is associated with increased probability or sequential treatment. Based on identified factors, multi-factorial model and on-line calculator was built for treatment prediction. The MRCCSECLINE calculator may become a practical tool to identify mRCC patients, who are unlikely to receive second-line treatment, and, therefore, to help determine optimal first lime treatment to obtain best response and treatment safety.